1. Field of the Invention
The present invention relates to a laser microscope for use particularly in biological, medical, and other applications, which irradiates a sample with a laser light constituted of a plurality of emission wavelengths through an objective lens and detects fluorescent light from the sample.
2. Description of the Related Art
There is a laser microscope for use in biological, medical, and other applications. In the laser microscope, for example, it is requested to observe a live cell or tissue over a long time as it is.
For example, there is an observation of a change of a concentration of calcium in the cell or the tissue. A method of the observation comprises: dyeing a sample with a fluorescent indicator which emits a fluorescent light in accordance with the calcium concentration; irradiating the sample with a laser light (excitation light) of a wavelength suitable for the fluorescent indicator; and detecting the fluorescent light from the sample.
In this case, the change of a signal (fluorescent light) from the cell or the tissue is generally remarkably small. Therefore, an intensity of the laser light with which the sample is irradiated is required to be stable at a high precision over a long time.
Some causes for which the intensity of the laser light for the irradiation of the sample is not stable are considered. There is a method of controlling the intensity of the laser light, comprising: monitoring the intensity of an emitted laser light; feeding the laser light intensity back to a controller; and controlling the laser light intensity. However, such feedback is not performed in a general helium neon laser. Therefore, an output power of the helium neon laser fluctuates with a change of environmental temperature, and the like.
Moreover, there is a multi-wavelength oscillation. For example, argon lasers oscillate with the laser light of wavelengths of 488 nm, 514.5 nm. Some of the argon lasers monitor and feedback-control the intensity of the emitted laser light.
However, the argon laser monitors a general output of the argon laser light of the wavelengths of 488 nm, 514.5 nm. The outputs of respective lines of these wavelengths compete among emission modes (wavelengths of 488 nm, 514.5 nm), and thereby each emission wavelength fluctuates. Furthermore, by consumption of an argon gas, an intensity ratio of the emission outputs of the argon laser light (intensity ratio of the wavelengths of 488 nm, 514.5 nm) changes with a use time.
On the other hand, there is a laser microscope for introducing the laser light into an optical fiber and guiding the laser light into a laser microscope main body by the optical fiber. In the laser microscope, the intensity of the laser light with which the sample is irradiated fluctuates by an output fluctuation by the optical fiber during undergoing of the change of environmental temperature, and a fluctuation of a light introduction efficiency by thermal deformation of a constituting element.
The intensity of the laser light fluctuates by the aforementioned causes, although the signal (fluorescent light) from the sample does not actually change. In this case, an erroneous result is possibly caused as if there were the change of the signal.
A technique for stabilizing the intensity of the laser light with which the sample is irradiated is disclosed, for example, in Jpn. Pat. Appln. KOKAT Publication Nos. 11-231222 and 2000-206415. In the Jpn. Pat. Appln. KOKAT Publication No. 11-231222, after the laser lights of a plurality of wavelengths are combined, some of the laser lights are split by a beam splitter. Subsequently, a changeable filter selects the wavelength, and an optical detector (first detection element) receives the laser light of the selected wavelength. Moreover, a laser output or a laser intensity is controlled based on a detection signal of the laser light intensity. It is described that the laser intensity is controlled, for example, by an acousto-optical element (e.g., an acousto-optical tunable filter (AOTF)) disposed between the laser and the optical fiber.
The Jpn. Pat. Appln. KOKAI Publication No. 2000-206415 discloses a method comprising: controlling an operation in combination with a linear filter ring driven by a control unit, an area selection filter ring, or a filter slider; detecting an output of a selected laser line; driving the AOTF based on the detection signal; and stabilizing the output of the selected laser line, in order to constantly monitor laser radiation connected to a scanning module.
In recent years, in order to further pursue a function of the cell or the tissue, it has strongly been requested to simultaneously detect two or more types of samples (fluorescent light) from the sample, and analyze the function. For example, for fluorescent proteins of different wavelengths, such as a green fluorescent protein (GFP: a protein emitting a green fluorescent light) and a red fluorescent protein (RFP: a protein emitting a red fluorescent light), a gene is developed in the cell, and observed with time.
In this case, the laser light with which the sample is to be irradiated needs to have a wavelength optimum for these fluorescent proteins GFP, RFP. Additionally, both the laser lights of the two wavelengths need to have the light intensities stabilized.
However, in the techniques described in the two publications, only the intensity of the laser light of one wavelength is stabilized, and the laser lights of two or more wavelengths cannot simultaneously be controlled so as to stabilize the intensities of the laser lights.
To solve the problem, an object of the present invention is to provide a laser microscope capable of simultaneously and steadily controlling an intensity of a laser light constituted of a plurality of wavelengths with which a sample is to be irradiated for each wavelength.